Response of the mouse uterus to nafoxidine stimulation: agonism and antagonism

Nafoxidine (NAF) acts as an estrogen agonist or antagonist depending on the animal model used. In the CD-1 mouse uterus, a three-day uterine bioassay of NAF produced a bell-shaped dose response curve with a maximal uterine wet weight increase at 200 micrograms/kg; this dose produced only a fractional increase in uterine dry weight. Combination treatment with NAF and estradiol antagonized estradiol stimulation of both wet and dry weight parameters. The time course of uterine wet weight stimulation following a single injection of NAF had an early pattern (0-10 h) similar to that of estradiol. However, at later times after stimulation, the patterns changed dramatically: the low NAF dose (200 micrograms/kg) returned to control levels by 24 h; estradiol and the high dose NAF (1.7 mg/kg) showed sustained stimulation, which peaked at 36 h with NAF compared to 24 h for estradiol. Nuclear estrogen receptor (ER) levels were measured after a single injection of 1.7 mg/kg NAF and showed a bimodal pattern similar to that seen with estradiol, with increases at 1 h and 8 h, although the overall ER levels were elevated above those seen with estradiol. Cytosolic ER levels with NAF decreased by 1 h and remained low up to 48 h. NAF treatment did stimulate uterine DNA and RNA synthesis, with a delayed time course compared to estradiol. DNA synthesis following a single 1.7 mg/kg dose of NAF was 2.5 times higher than that produced by 20 micrograms/kg estradiol. NAF treatment resulted in hypertrophy and hyperplasia in the luminal epithelium but not in the glandular epithelium. Long-term exposure to estradiol for 5 wk resulted in development of uterine cystic glandular hyperplasia and increased secretory activity; long-term exposure to NAF produced a more significant tissue hyperplasia but no secretions. These studies show that NAF stimulates some of the receptor-mediated responses attributed to an estrogen agonist in the mouse uterus; but, when co-administered with estradiol, NAF antagonizes some aspects of estrogen action.     

Reproductive Functions Illustrating Direct and Indirect Effects of Genes on Behavior

Effects of gene products on reproductive behavior which are relatively direct include those of the estrogen receptor and progesterone receptor. For example, work with estrogen receptor-deficient (ERKO) female mice has extended previous evidence contributing to the neurochemical analysis of lordosis behavior. On the other hand, sex differences in behavior present a classic example of indirect effects of genes on behavior. Work with ERKO male mice shows the necessity of ER gene expression for normal masculinization of the brain. In particular, behavioral assay results distinguish apparent motivational performance of ERKO males from male mating reflexes: the former is similar to that of wild-type males in important respects, while the latter are deficient in ERKO males. The present paper first reviews a small number of clear genetic contributions to reproductive behaviors, and then reports one experiment pertinent to the interpretation of the behavioral status of ERKO male mice.     

A novel fluorinated derivative of diethylstilbestrol.

The synthesis of the diethylstilbestrol (DES) derivative with fluorine atoms present in the positions ortho to the hydroxyl in each ring is described. In vitro studies in a system containing horse radish peroxidase/H2O2 demonstrate extensive oxidation of tetrafluorodiethylstilbestrol to the corresponding dienestrol derivative. Tetrafluorodiethylstilbestrol and DES had comparable in vivo uterotropic activities at a dose of 100 microgram/kg. Competitive binding experiments demonstrated 20-25 fold reduced interaction with the mouse uterine estrogen receptor. This compound may be useful as an experimental estrogen in distinguishing between the biological and toxic effects of DES.     

Characterization of murine cell lines from Diethylstilbestrol-Induced uterine endometrial Adenocarcinomas

Neonatal treatment with estrogens is associated with development of uterine adenocarcinomas in CD-1 mice. Treatment with the synthetic estrogen diethylstilbestrol (DES) on Days 1 to 5 after birth results in 90% incidence of these hormonedependent lesions in 18-mo.-old mice. Three cell lines were established from these DES-associated tumors. Each of these cell lines exhibited morphologic and ultrastructural characteristics of transformed epithelial cells, including an increased nuclear:ytoplasmic ratio, enlarged and irregular nuclei with multiple nucleoli and areas of chromatin condensation, positive staining for cytokeratin, desmosomes, and microvilli. After subcutaneous injection into nude mice, all three cell lines formed solid tumors within 4 wk. Although the primary uterine tumors and tumor transplants in nude mice had been shown to be estrogen-dependent and estrogen-receptor positive, neither the monolayer growth nor the tumorigenicity of any of the three cell lines in this study was enhanced by or dependent on estrogen. Estrogen receptor levels were low in early and intermediate passage cells. Allele-specific oligonucleotide hybridization analysis of PCR-amplified cell line DNA revealed no point mutations in the 12th, 13th, or 61st codons of the K-ras or H-ras protooncogenes. Southern analysis revealed no changes in genomic organization of the putative tumor suppressor gene DCC, but demonstrated a three-to four-fold amplification of the c-myc gene in one cell line. Expression of c-myc RNA was concomitantly increased in the same cell line. These three transformed cell lines represent the end point in the process of hormone-associated tumorigenesis and as such should prove useful in investigating the molecular changes and the mechanisms involved in hormonal carcinogenesis.     

Oxytocin and estrogen receptor alpha and beta knockout mice provide discriminably different odor cues in behavioral assays

Social behavior involves both the recognition and production of social cues. Mice with selective deletion (knockout) of either the gene for oxytocin (OT) or genes for the estrogen receptor (ER) -α or -β display impaired social recognition. In this study we demonstrate that these gene knockout mice also provide discriminably different social stimuli in behavioral assays. In an odor choice test, which is a measure of social interest and discrimination, outbred female Swiss-Webster mice discriminated the urine odors of male knockouts (KO: OTKO, αERKO, βERKO) from the odors of their wildtype littermates (WT: OTWT, αERWT, βERWT). Females showed marked initial choices of the urine odors of OTWT and βERWT males over those of OTKO and βERKO males, and αERKO males over αERWT males. The odors of OTKO and βERKO males also induced aversive, analgesic responses, with the odors of WTs having no significant effects. Odors of both the αERWT and αERKO males induced aversive, analgesic responses, with the odors of the WT inducing significantly greater analgesia. The odors of restraint stressed WT and KO males also elicited analgesia with, again, females displaying significantly greater responses to the odors of stressed OTKO and βERKO males than their WTs, and significantly lower analgesia to the odors of stressed αERKO than αERWT males. These findings show that the KO mice are discriminated from their WTs on the basis of odor and that the various KOs differ in the relative attractiveness/aversiveness of their odors. Therefore, in behavioral assays one causal route by which gene inactivation alters the social behavior of knockout mice may be mediated through the partners' modified responses to their odors.     

STING Millennium Suite: integrated software for extensive analyses of 3d structures of proteins and their complexes

Background  

The integration of many aspects of protein/DNA structure analysis is an important requirement for software products in general area of structural bioinformatics. In fact, there are too few software packages on the internet which can be described as successful in this respect. We might say that what is still missing is publicly available, web based software for interactive analysis of the sequence/structure/function of proteins and their complexes with DNA and ligands. Some of existing software packages do have certain level of integration and do offer analysis of several structure related parameters, however not to the extent generally demanded by a user.     

Genome and Hormones: Gender Differences in Physiology: Selected Contribution: Cerebrovascular NOS and cyclooxygenase are unaffected by estrogen in mice lacking estrogen receptor-alpha

Estrogen alters reactivity of cerebral arteries by modifyingproduction of endothelium-dependent vasodilators. Estrogen receptors (ER) are thought to be involved, but the responsible ER subtype isunknown. ER- knockout (ERKO) mice were used to test whether estrogen acts via ER-. Mice were ovariectomized, with or without estrogen replacement, and cerebral blood vessels were isolated 1 molater. Estrogen increased levels of endothelial nitric oxide synthaseand cyclooxygenase-1 in vessels from wild-type mice but was ineffectivein ERKO mice. Endothelium-denuded middle cerebral artery segmentsfrom all animals constricted when pressurized. In denuded arteries fromERKO but not wild-type mice, estrogen treatment enhancedconstriction. In endothelium-intact, pressurized arteries fromwild-type estrogen-treated mice, diameters were larger compared witharteries from untreated wild-type mice. In addition, contractileresponses to indomethacin were greater in arteries from wild-typeestrogen-treated mice compared with arteries from untreated wild-typemice. In contrast, estrogen treatment of ERKO mice had no effect ondiameter or indomethacin responses of endothelium-intact arteries. ThusER- regulation of endothelial nitric oxide synthase andcyclooxygenase-1 pathways appears to contribute to effects of estrogenon cerebral artery reactivity.

     

Genotype/age interactions on aggressive behavior in gonadally intact estrogen receptor beta knockout (betaERKO) male mice

Estrogen, as an aromatized metabolite of testosterone, has a facilitatory effect on male aggressive behavior in mice. Two subtypes of estrogen receptors, alpha (ER-alpha) and beta (ER-beta), in the brain are known to bind estrogen. Previous studies revealed that the lack of ER-alpha gene severely reduced the induction of male aggressive behavior. In contrast, mice that lacked the ER-beta gene tended to be more aggressive than wild type (WT) control mice, although the behavioral effects of ER-beta gene disruption were dependent on their social experience. These findings lead us to hypothesize that estrogen may facilitate aggression via ER-alpha whereas it may inhibit aggression via ER-beta. In the present study, we further investigated the role of ER-beta in the regulation of aggressive behavior by examining developmental changes starting at the time of first onset, around the age of puberty. Aggressive behaviors of ER-beta gene knockout (betaERKO) mice were examined in three different age groups, puberty, young-adult, and adult. Each mouse was tested every other day for three times in a resident-intruder paradigm against olfactory bulbectomized intruder mice and their trunk blood was collected for measurements of serum testosterone after the completion of the study. Overall, betaERKO mice were significantly more aggressive than WT. These genotype differences were more pronounced in puberty and young adult age groups, but not apparent in the adult age group, in which betaERKO mice were less aggressive than those in two younger age groups. Serum testosterone levels of betaERKO mice were significantly higher than those of WT mice only in the pubertal age group, but not in young adult (when betaERKO mice were still significantly more aggressive than WT mice) and adult (when no genotype differences in aggression were found) age groups. These results suggest that ER-beta mediated actions of gonadal steroids may more profoundly be involved in the inhibitory regulation of aggressive behavior in pubertal and young adult mice.